Preparation containing cell extracts for synthesizing cell-free protein and means for synthesizing cell-free protein

ABSTRACT

Disclosed are a preparation containing cell extracts for cell-free protein synthesis, prepared by excluding from a living organism a system, participating to inhibiting of self protein synthesis reaction, an apparatus for cell-free protein synthesis reaction equipped with a reaction tank for cell-free protein synthesis, and a kit for use therefor; the preparation can be stored at room temperature and prepared as a preparation in a state where biological functions of the cell extracts are maintained and further, disclosed is means for cell-free protein synthesis comprising cell extracts from which an inhibitor for self protein synthesis reaction is substantially excluded, having introduced therein treatment selected from supplement, storage, exchange or discharge with respect to an element selected from at least mRNA serving as a template for synthesis reaction, an energy reproduction system enzyme, a substrate, and an energy source.

The present application is a 371 of PCT/JP99/04088, filed Jul. 29, 1999.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a preparation containing a cellextracts for cell-free protein synthesis prepared from cells and tissuesfor use in cell-free protein synthesis system, the preparation obtainedby stabilizing thereof, enabling of storing and transporting at roomtemperature, and means and apparatus for synthesizing the cell-freeprotein using the same.

BACKGROUND OF THE INVENTION

Protein synthesizing reactions which occur in cells proceed in a processto synthesize the protein in which first from DNA having geneticinformation, the information is transcribed to mRNA and ribosomestranslate said information of mRNA. Currently, as the method forperforming ex vivo protein synthesis, e.g., in vitro, there has beenintensively conducted investigation on a cell-free protein synthesizingmethod in which ribosomes are extracted from a living organism and invitro protein synthesis is performed using thereof (Japanese PatentLaid-open Publication Nos. Hei 6-98790, Hei 6-225783, Hei 7-194, Hei9-291, and Hei 7-147992). In this method, Escherichia coli, embryo,rabbit reticulocyte, etc. have been used as a raw material of ribosome.

Cell-free protein synthesizing reaction mixture containing cell extractsfor use in cell-free protein synthesizing system and chemical substanceswhich are indispensable for or increases an efficiency of translationreaction, such as other synthetic substrates excluding translationtemperate, energy sources and various ions, and etc. are instable at anordinary temperature and their stable storage has been merely possibleat a super low temperature of −80° C. or lower.

Cell-free protein synthesizing system is a useful method which iscapable of retaining accurate performances, comparable to living cellsin a peptide synthesis reaction rate and translation reaction andobtaining target protein without practicing complicated purificationstep. Therefore, to more usefully apply the synthesizing system inindustry, several inventions relating to an improvement in synthesizingefficiency has been published. However, in order to improve theusefulness in industry, it is necessary to provide not only synthesizingefficiency but also various substances used in the synthesizing systemwith stably retaining and supplying in high quality.

An object of the present invention is to provide a means capable ofbeing stable in an ordinary temperature and maintaining biologicalfunction of the preparation containing a cell extracts for cell-freeprotein synthesis containing cell extracts for cell-free proteinsynthesis necessary for cell-free protein synthesizing system, andchemical substances, which is indispensable for or increases theefficiency of the translation reaction, such as other synthesizingsubstrates excluding translation templates, energy sources, and variousions, etc.

Another object of the present invention is to provide a means for stablystoring and supplying a kit comprising a preparation containing cellextracts for cell-free protein synthesis, thereby simplifying operationsteps in cell-free protein synthesis.

Still another object of the present invention is to provide a means forcell-free protein synthesis using a preparation containing cell extractsfor cell-free protein synthesis, which is improved in productivity,yield, and simplicity.

SUMMARY OF THE INVENTION

The inventors of the present invention have made intensive investigationto solve above objects and, as a result, the inventors completed thepresent invention by, as one of means, excluding a system participatingin inhibiting protein synthesis reaction, per se, in cells for cell-freeprotein synthesis as a raw material. Another means of the presentinvention includes application of a treatment such as freeze-drying cellextracts prepared for cell-free protein synthesis, or cell extracts forcell-free protein synthesis and substances participating in cell-freeprotein synthesizing reaction system, to prepare a dry preparationthereby completing the present invention.

Further, still another means of the present invention includes, as acell-free protein synthesizing method using cell-free proteinsynthesizing system obtained by the above means, provision of cell-freeprotein synthesis means applying a principle of molecular sieving.

Yet another cell-free protein synthesis means is a continuous cell-freeprotein synthesizing means with applying a dialysis membrane and relatesto additional introduction of selected elements and to apparatustherefor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows protein synthesis activity in which freeze-dried wheat germextract stored at −80° C. for 2 months comparing with non-freeze-driedwheat germ extract stored in liquid nitrogen for 2 months. In thefigure, symbols indicate extract stored in liquid nitrogen for 2 monthsaccording to a conventional method as (□-□) and one stored for the sameperiod of time after freeze-drying as (▪-▪), respectively, and ordinaterepresents radioactivity incorporated in protein (DPM/5 μl of reactionmixture) and abscea represents reaction time at 26° C.

FIG. 2 shows an effect of storage temperature for freeze-dried extracton the protein synthesis activity. In the figure, symbols indicateextract stored at room temperature for 1 month as (□--□), one stored at4° C. for the same period of time as (▪-▪), and one stored at −80° C.for the same period of time as (●--●), respectively.

FIG. 3 shows the protein synthesis activity of synthesis solution forfreeze-dried wheat germ cell-free protein in a batch reaction system. Inthe figure, symbols indicate extract stored in liquid nitrogen for 2months according to a conventional method as (▪-▪) and one stored forthe same period of time after freeze-drying as (□-□), respectively.

FIG. 4A is a photograph illustrating synthesizing results of cell-freeprotein utilizing an open column. Lane 1 shows results of absence oftemplate and Lane 3 shows results of synthesis for 12 hours by a columnmethod. Lane 2 shows, as a comparative example, results of synthesis forthe same period of time by a continuous cell-free protein synthesismethod using a conventional dialysis method. In FIG. 4A, the left-sideLane represents molecular weight markers of 94 kDa, 67 kDa, 43 kDa, 30kDa, and 20.1 kDa, respectively, from top. Arrows indicate synthesizeddihydrofolate reductase.

FIG. 4B is a photograph illustrating synthesizing results of cell-freeprotein utilizing a commercially available liquid chromatographyapparatus. Lane 1 shows, as a comparative example, results of synthesisfor 12 hours by a continuous cell-free protein synthesis method using aconventional dialysis method. Lane 2 shows, as a comparative example,synthesis results for 12 hours by a continuous cell-free proteinsynthesis method using a conventional dialysis method. Lane 2 showssynthesis results for the same period of time by a cell-free proteinsynthesis method using a liquid chromatography method. Arrows indicatesynthesized dihydrofolate reductase.

FIG. 5 is a perspective view showing a cartridge apparatus.

FIG. 6 is a central cross-sectional view showing a cartridge apparatus.

In FIGS. 5 and 6, each symbol has the following meaning.

-   1 Impregnation tank-   2 Lid portion-   3 Dialysis external liquid-   4 Liquid level of dialysis external liquid-   5 Inlet for supplying substrate and/or energy source, etc.-   6 Outlet in the impregnation tank for supplying substrate and/or    energy source, etc.-   7 Channel-   8 Inlet in an impregnation tank for discharging a dialysis external    liquid-   9 External discharge outlet for discharging a dialysis external    liquid-   9 a External discharge outlet for discharging a dialysis external    liquid-   10 Channel-   11 Inlet for supplying mRNA and/or energy reproduction system enzyme-   12 Medium having a function of dialysis membrane-   13 Introductory part for Inlet 11-   14 Jig for holding a membrane-   15 End portion of a membrane-   16 Magnetic stirrer-   17 Introductory part for inlet 5-   18 Introductory part for outlet 6 in an impregnation tank-   19 Introductory part for inlet 8 in an impregnation tank-   20 Introductory part for external discharge outlet 9-   20 a Introductory part for external discharge outlet 9 a

FIG. 7 shows maintenance of the effect of protein synthesis supplementaladdition of mRNA and creatine kinase. In the figure, o-o indicatessupplemental addition of mRNA coding for dihydrofolate reductase with aCAP and creatine kinase, Δ-Δ indicates supplemental addition of mRNAonly, ∇-∇ indicates supplemental addition of creatine kinase only, □-□indicates no supplemental addition, and arrows indicate timing ofsupplemental addition.

FIG. 8 shows maintenance of the effect for protein synthesis by exchangeof dialysis external liquid, with o-o indicating that exchange ofdialysis external liquid has been performed, Δ-Δ indicating that noexchange has been performed, and arrows indicating timing of exchange.

FIGS. 9A, 9B and 9C show maintenance of the effect for protein synthesisby use of an automatic apparatus. In the figure, o-o indicatessupplemental addition of mRNA coding for dihydrofolate reductase with aCAP and creatine kinase, Δ-Δ indicates no supplemental addition of mRNA,white arrows indicate dihydrofolate reductase, synthesis product, andsymbol (*) s indicate dyed band of the supplemented creatinine kinase.

BEST MODE FOR CARRYING OUT THE INVENTION

1) Exclusion of a System Participating in Inhibition of Own ProteinSynthesizing Reaction in Cell for Cell-Free Protein Synthesis

Exclusion of a system participating in inhibition of own proteinsynthesizing reaction in cell, as a raw material, for cell-free proteinsynthesis, as one of means of the present invention, signifies a removalof means for controlling the synthesis of the own protein that the rawmaterial cells themselves contain or hold therein. In particular,exclusion of the participating system that the present inventors havefound, signifies an exclusion of substances, which act on ribosome,translation protein factors, mRNA, and tRNA, and inhibit the functionsthereof.

The substances contained or held by raw material cells that suppress thefunction for protein synthesis include tritin (Massiah, A. J., andHartely, M. R. (1995) Planta, 197, 633-640), protein called thioninethat contains much cystein (Brummer, J., Thole, H. and Kloppstech, K.(1994) Eur. J. Biochem, 219, 425-433), ribonuclease (Matsushita S., Mem.Res. Inst. Food Sci. Kyoto Univ., No. 19, 1-), and the like, which havebeen known to be abundantly localized in, for example, an albumen of aseed.

By completely excluding from a germ specimen a group of cell-free germprotein synthesis suppressing (inhibiting) proteins localized in albumencontaminating in the step of isolating germ, e.g., tritin, thionine,ribonuclease, etc., inactivation reaction in protein synthesis can becanceled.

That is, one of the means of the present invention can be achieved by atechnology for removing self protein synthesizing reaction suppressingmechanism that operates when a tissue of a living organism or cells areinjured, that is, self protein synthesizing reaction destructingmechanism as an anti-pathogen self defensive mechanism physiologicallyfurnished, and a technology for neutralizing or removing proteinsynthesis reaction suppressing activity that is induced upon thedestruction, and the preparation of extracts for cell-free proteinsynthesis from cells or tissue.

As the raw material cell of the present invention, there can bebasically used germs, Escherichia coli, reticulocytes, cancer cells,etc., which are used generally in cell-free protein synthesizingsystems, and in addition, cells for cell-free protein synthesisoriginating from other organisms. In a preferred embodiment, the rawmaterial cell includes germs originating, for example, wheat, barley,rice plant, corn, spinach, buckwheat, and etc.

Preparation of cell extracts for cell-free protein synthesis of thepresent invention from these raw material cells may be performed incombination with various known methods (Johnston, F. B. et al. (1957)Nature, 179, 160-161). The means useful for removing the means forcontrolling in synthesis of self proteins, which is one of the presentinventions, is to treat the raw material cells with a surfactant, inparticular a nonionic surfactant. A wide variety of nonionic surfactantsmay be used so far as it is a nonionic. Preferred examples thereofinclude Brij, Triton, Nonidet P-40, Tween, and the like, which arepolyoxyethylene derivatives. Among them, Nonidet P-40 is the mostpreferred. These nonionic surfactants are used in a concentration of,for example, 0.5%.

When wheat germ, for example, is used as raw material cell, thetreatment is to recover germ extracts by steps for milling, floatatingand sieving using known means. The germ extracts are washed with asurfactant several times in order to exclude the albumen portion whichhas contaminated therein. This is performed until the washings are notturbid. The treatment is carried out preferably in combination withultrasonication, which gives rise to a more complete effect.

The germ extracts, thus obtained, are substantially completely free fromalbumen (endosperm) that contains an endogenous specific inhibitor, suchas tritin, and etc., and the germ has been purified substantially. Uponassaying the contamination of albumen proteins before and after theultrasonication by an immunoblot method using an anti-tritin antibodyand tritin as an index, it has been confirmed that ultrasonicationwashing resulted in reducing tritin content to below detection limits.This indicates that the germ extracts is substantially free fromcontamination with albumen proteins and that other protein synthesisinactivating factors localized in albumen, such as thionine andribonuclease, are also removed from the germ extracts. The obtained germextracts are purified to such an extent that ribosomes are notdeadenylated substantially, and the ribosome deadenylation ratio isbelow 7%, preferably 1% or less.

2) Means for Stabilizing Preparation

Another means of the present invention is to introduce for stabilizingmeans preparation for the thus-prepared cell extracts for cell-freeprotein synthesis. Hitherto, the method for storing the cell extractsfor cell-free protein synthesis is to store in the vicinity of −80 to−196° C., while by introducing the means, a preparation containing thecell extracts for cell-free protein synthesis can be stored stably atroom temperature. Achievement of the technology in which the preparationof the present invention can be stored at room temperature, is useful inindustry.

The means for stabilizing the cell extracts for cell-free proteinsynthesis is to form a preparation by means of dry process, inparticular by means of freeze-drying. The freeze-drying may be performedby using a method known, per se. For example, the cell extracts forcell-free protein synthesis are quickly frozen with liquid nitrogen. Thedrying is performed for 3 hours using a conventional freeze-dryingapparatus. After completion of the removal of water, the obtainedpowdery preparation is sealed under vacuum or nitrogen atmosphere, thusbeing capable of formating into the preparation.

The above preparation may be formed with only the cell extracts forcell-free protein synthesis that have been treated by the means of thepresent invention, but if desired, may be formed into a preparationafter selection and addition of substances essential in the cell-freeprotein synthesizing system, for example, synthesized substrates, aminoacids or energy sources.

To the above preparation may be added substances which increases thereaction efficiency of cell-free protein synthesizing system, forexample, various ionic compounds, preferably potassium ion compound,magnesium ion compound, etc.

Further, to the preparation may, if desired, be added substances whichenhances solubility, for example, surfactants, substances which protectsthe above ribosomes from deadenylation thereof.

More preferably, the preparation is adjusted to a composition which mayachieve optimization of the reaction by merely adding water. Thecomposition may be used by forming a kit of the preparation and mixingthereof on demand. Of course, in the formation of a kit, cell extractsfor cell-free protein synthesis alone, substances essential to cell-freeprotein synthesizing system, substances which increase the reactionefficiency of cell-free protein synthesizing system, and further aqueoussolution suitable for the optimization of the reaction, etc. may beselected for forming the kit.

3) (Means for Cell-Free Protein Synthesis: Utilization of a MolecularSieve Carrier)

In another embodiment of the present invention, a reaction tank for usein cell-free protein synthesis systems may be prepared with a carriercapable of molecular sieving. The carrier is not limited, particularlyso long as it is suitable for a fractionation molecular weight of 10,000or less. The carrier material includes, for example, porous gelfiltration particles, more specifically Sephadex G10 to G25, etc. Thecarrier may be adjusted with water. More preferably the carrier isequilibrated with a buffer solution before use. The carrier may beadjusted after filling in a reaction tank or may be formed into a kitseparately from the reaction tank, so that it may be adjusted on demand.

The reaction tank is preferably one which allows chromatography, morepreferably column chromatography. The column diameter, column length,and carrier volume can be adjusted appropriately by combination ofreaction time and developing speed. The volume of reaction tank can beselected appropriately depending on an amount of synthesis of targetprotein. The suitable reaction tank is one having at least two openingsthat can be opened when needed, so that a carrier may be filled inadvance and is adjusted so that raw material substances participating inthe cell-free protein synthesis system may be dispersed therein, ifdesired.

The filling of the carrier in the reaction tank is possible when neededor in advance. It is desirable that the raw material substancesparticipating in the cell-free protein synthesizing system, inparticular synthesis substrates, for example, amino acids, energysources, including ATP, GTP, creatine phosphate, and ionic componentsthat increase the efficiency for cell-free protein synthesizing reactionthat are added, if desired, be dispersed uniformly in the filledcarrier, more preferably in an optimum dispersion state, particularly ina localized uniform dispersion. The dispersion state is attained withcalculating reaction efficiency in taking consideration of anassociation of development of these substances in the mobile phase ofthe carrier and of a development speed of the cell extracts forcell-free protein synthesis and/or translation template substance,separately filled.

Here, if desired, the carrier may be exclusively constituted with amolecular sieve carrier and separately a preparation which is preparedin advance with appropriately selected cell extracts for cell-freeprotein synthesis, synthesis substrates, energy sources, and if desired,ionic components that increase the efficiency for cell-free proteinsynthesizing reaction, is filled into the carrier on demand, and atranslation template substance is filled and developed, therebyperforming the cell-free protein synthesis.

The development speed of a mobile phase in the carrier capable ofmolecular sieving depends, on the size of a column and synthesisefficiency, but generally, the speed ranges from {fraction (1/10)} to{fraction (1/30)} of an inner volume of the column per hour. Thesolution used for development is a solution which contains a synthesissubstrate, for example, amino acid, an energy source including ATP, GTPand creatine phosphate, and ionic components optionally added toincrease the efficiency for cell-free protein synthesizing reaction, butit is not limited thereto. The development is preferably controlledautomatically but it is not limited thereto.

The synthesizing reaction is preferably carried out in a reaction tankfilled with the carrier. However, in consideration of the fillingconditions, the reaction may be conducted in the carrier supernatant, ormay be started in a batch, further followed by synthesizing the reactionduring carrier development.

The synthesis reaction of the present invention is characterized in thatthe cell extracts for cell-free protein synthesis, such as ribosomes,move as a mobile phase, and by-products generated in the synthesizingreaction are separated and removed by fractionation-wise development,and that at the same time, the synthesis proceeds at the maximumreaction rate under supplying chemical substances which areindispensable for translation reaction, or for increasing the reactionefficiency, such as synthesis substrates, energy sources, and variousions at optimum concentrations.

The means of the present invention can be practiced by automaticallycontrolled filling and developing material substances participating incell-free protein synthesis system and recovering synthesized protein.The automation may be controlled by every process unit, if desired. Morepreferably, the solution development speed in mobile phase isautomatically controlled.

The means of the present invention can continuously recover a largeamount of protein in a high purity by a linkage of purification meansknown per se, such as various ion exchange columns or affinity columns,and etc., depending on the target protein.

Thus, by use of the means of the present invention explained above, amethod for efficiently producing the target protein can be provided.

Further, by use of the means of the present invention explained above,an apparatus for the target cell-free protein synthesis can be provided.

In addition, by use of the means of the present invention explainedabove, a kit comprising a set suitable for the target cell-free proteinsynthesis can be provided.

4) Means for Continuous Cell-Free Protein Synthesis (SupplementalAddition of Each Element)

In another embodiment, the present invention can be achieved bysupplemental addition of each element for use in cell-free proteinsynthesis system.

In the present invention, mRNA serving as a template for synthesisreaction is supplemented on demand or continuously after initiating thereaction, at around a time for appearing a tendency in which thetemplate activity of mRNA added as a raw material decreases. Theaddition may be made on demand in a very small amount continually, orperiodically. The addition amount is an order of from one tenth toequivalent to the amount of raw material mRNA. The feature of thepresent invention is that the effect of supplemental addition has beenconfirmed for the first time and the addition amount and timing can bereadily changed or fixed by those skilled in the art with confirming theeffect of synthesis. The same is true for other elements and in the samesense, even if there will be no specific description hereinbelow.

In the present invention, an enzyme in an energy reproduction system issupplemented on demand or continuously after initiating the reaction, ataround a time for appearing a tendency, in which the activity of energyreproduction system enzyme added as a raw material decreases. Theaddition may be made in a very small amount continually or periodically.The addition amount is an order of from one tenth to equivalent to theamount of raw material energy reproduction system enzyme. The energyreproduction system enzyme is suitably creatine kinase. Of course,substances having similar function to that of the present invention cansimilarly be added as an enzyme for an energy reproduction system. Theaddition amount and method therefor can be changed on demand, withreferring to the amount of synthesis, as a marker.

The supplemental additions of mRNA and the enzyme for the energyreproduction system may be performed separately each other, but maypreferably be made in combination. The addition method may be eithercontinuously or intermittently. The addition amount and the methodtherefor may be changed on demand with referring to the amount ofsynthesis, as a marker.

In the present invention, in addition to the supplemental addition ofmRNA and/or the supplemental addition of enzyme for energy reproductionsystem, a step for preventing the exhaustion of substrate and/or, energysource and/or a step for discharging by-products can be accompanied. Itis preferred that various amino acids, ATP, GTP, etc. are supplementallyadded as a substrate or energy source continuously or intermittently.The addition amounts thereof are preferably maintained at concentrationsof respective substances at the time for initiating synthesis or atconcentrations close thereto. However, the addition amounts may besupplemented or changed when needed using the effect of synthesis as amarker.

A discharge of the by-products means discharging metabolites such as AMPand GMP, etc., and reaction products, such as phosphoric acid andpyrophosphoric acid, etc., and such compounds are preferably dischargedfrom the reaction system continuously or intermittently.

As steps for preventing the exhaustion of substrate and/or energysource, and/or steps for discharging by-products are/is preferablycontinuous or intermittent renewal of the reaction medium in thereaction system. In the present invention, for example, a method inwhich a dialysis membrane is used is cited. In this case, for example, adialysis external liquid is continuously or intermittently renewed orexchanged.

The supplement, storage, exchange or discharge of each element aretreated preferably in automation. The means for automation is to providean apparatus under a control of computer system known, per se., andperforms the supplement, storage, exchange or discharge of each elementintegrally. Reagents for use in these steps as respective elements arepreferably prepared in the form of a kit.

5) Apparatus for Automatic Continuous Cell-Free Protein Synthesis

An apparatus for automatic cell-free protein synthesis is under acontrol of computer system known, per se., and has, in combination, afunction for setting up an environment optimal for protein synthesis, inaddition to the function for integrally performing the supplement,storage, exchange or discharging of each element.

That is, a plurality of dialysis vessels can be set at a desiredtemperature by placing, for example, in a plurality of chambersindependent of each other, each equipped with an electronic coolingapparatus and a heater and being capable of varying temperature rangingfrom 15 to 37° C., thereby being capable of maintaining the optimaltemperature for the synthesis of the target protein.

As the method for continuously or intermittently renewing or exchangingthe dialysis external liquid in a dialysis vessel, the rate of renewalor exchange of dialysis external liquid can be varied between 0.1 and 1ml/hour, for example, by use of a dispensing apparatus such as aperistaltic pump or a syringe pump, etc., continuously or intermittentlyto thereby select optimal conditions for the synthesis of the targetprotein.

Further, substances necessary for the protein synthesis, such astemplate mRNA and creatine kinase, an energy reproduction system enzyme,etc., may be supplementally added in a desired amount to a proteinsynthesizing reaction system portion placed in the plurality of dialysisvessels for every desired times, for example, at an interval of 6 to 15hours.

In the above apparatus, respective elements such as template mRNA, asubstrate for an energy reproduction system enzyme, an energy source, adialysis external liquid, etc., are stored individually or in admixturein storage vessels connected to dialysis vessels, respectively, andsupplied through respective passages that connect the storage vessels tothe protein synthesizing reaction system portions or dialysis vessels.The storage vessels are maintained preferably at 4° C.

6) Constitution of an Apparatus for Continuous Cell-Free ProteinSynthesis

As an example of an apparatus for use in a continuous cell-free proteinsynthesis system for embodying the present invention, a cartridgeapparatus will be explained. However, the apparatus of the presentinvention does not have to be of a cartridge type.

The cartridge apparatus is an apparatus for continuous cell-free proteinsynthesis having a constitution comprising an impregnation tank which isgenerally of a hollow body with a bottom and lid portion fitted theretotightly sealably. This apparatus carries a passage having an inlet asmeans for introducing a substrate and/or an energy source and an outletcommunicating with a liquid chamber in the impregnation tank for thedialysis external liquid, a passage having an inlet existing in theliquid chamber in the impregnation tank, whose inlet is as means fordischarging metabolites, etc. in the dialysis external liquid and anoutlet communicating with outside, an inlet as means for introducingmRNA and/or an energy reproduction system enzyme, and a medium havingthe function of a dialysis membrane existing in the liquid chamber inthe impregnation tank for the dialysis external liquid. Hereafter, thecartridge of the present invention will be described in detail referringto the drawings. However, the apparatus shown in the drawings is a mereembodiment and the present invention is not limited thereto.

The cartridge apparatus includes an impregnation tank 1, which is of agenerally hollow body with a bottom, and a lid portion 2 fitted theretotightly and sealably and a dialysis external liquid 3 is filled in theimpregnation tank 1 up to a level 4. More specifically, the apparatuscarries as a fundamental constitution a passage 7 having an inlet 5 asmeans for introducing an energy source, etc. and an outlet 6communicating with a liquid chamber in the impregnation tank 1 for thedialysis external liquid 3, a passage 10 having an inlet 8 existing inthe liquid chamber in the impregnation tank 1, whose inlet is as meansfor discharging metabolites, etc. in the dialysis external liquid 3 andan outlet 9 and/or 9 a communicating with the outside, and an inlet 11as means for introducing substances for the synthesis system, and amedium 12 having the function of a dialysis membrane existing in theliquid chamber in the impregnation tank 1 for the dialysis externalliquid 3. As an embodiment, FIGS. 5 and 6 show a cartridge apparatuswhose impregnation tank is of a cylindrical body. However, the shape ofimpregnation tank is not limited to a cylindrical body.

Here, the outlet 6 communicates with the dialysis external liquid 3 inthe liquid and an end portion of the outlet 6 is positioned in the upperhalf of the impregnation tank 1 and at least below the liquid level 4.The inlet 8 communicates with the dialysis external liquid 3 in theliquid and an end portion of the inlet 8 is positioned in the lower halfof the impregnation tank 1. The position of the end portion of theoutlet 6 and the position of the end portion of the inlet 8 may beupside down.

The outlet 9 a and the introductory part 20 a contact with the liquidlevel 4 at the same site and are positioned at an upper position thanthe liquid level 4. The outlet 9 a and the introductory part 20 atogether enable discharge by spontaneous flowing out. In the case wheredischarge of the metabolites, etc. in the dialysis external liquid 3 isperformed through the outlet 9 and the introductory part 20, the outlet9 a and the introductory part 20 a may be closed or the impregnationtank molded without the outlet 9 a and the introductory part 20 a at thetime of molding may be used. In the case where the discharge of themetabolites, etc. in the dialysis external liquid 3 is performed byspontaneous discharge through the outlet 9 a and the introductory part20 a, the outlet 9 and the introductory part 20 may be closed or theimpregnation tank molded without the outlet 9 and the introductory part20 at the time of molding may be used. In this case, though not shown,the introductory part 19 and the introductory part 20 a may be moldedand used so that the introductory part 20 a of the outlet 9 a and theintroductory part 19 of the inlet 8 can be connected to each other. Uponthe molding, the end of introductory part 19 of the inlet 8 on the sideof the outlet 9 is not connected to the lid portion but is connectableto the introductory part 20 a of the outlet 9 a.

The medium 12 having the function of a dialysis membrane may be moldedintegrally with an introductory part 13 of the inlet 11 or fixed theretoby means of a jig 14 (Jig for holding a membrane) for holding themembrane as illustrated. The medium 12 is arranged in the impregnationtank 1 in such a position that it is immersed in the dialysis externalliquid 3. The end opposite to the direction for connecting to the inlet11 of the medium 12 may be arranged in the impregnation tank 1 in such aposition that it is immersed in the dialysis external liquid 3 and it issufficient if it is closed. As a specific example thereof, it may beclosed a lid-like member such as a membrane end portion 15 or the medium12 may be of a bag-like structure having only one opening.

The cartridge apparatus may preferably have means for stirring thedialysis external liquid 3. As a specific example, a rotary medium suchas a magnetic stirrer 16 may be placed in the impregnation tank 1.

The impregnation tank 1 is preferably made homeostatic for the synthesisefficiency and may be used in combination with a desired homeostaticmeans. For example, the impregnation tank 1 may be adjusted so that itcan be arranged in an incubation tank. The cartridge apparatus isprovided to users usually in a state where the dialysis external liquid3 and synthesis system substances are not immersed and users flow theliquid through the inlet 11 and the inlet 5 in any time desired.

The inlet 5, the outlet 9 and the inlet 11 may have means for automaticcontrol for flow in/out therethrough. As easier automation means, it ispreferred that the flow in/out system from the inlet 5 to the outlet 6and from the inlet 8 to the outlet 9 is automatically controlled so asto maintain the liquid level 4 at a constant level. In this case, thesupplemental addition of a substance through the inlet 11 may beperformed by manual control. In the case where the outlet 9 a and theintroductory part 20 a are arranged at a site above the liquid level 4and contacting the liquid level, spontaneous flowing out is possible.

The cartridge apparatus of the present invention may be pre-assemblingapparatus or may be one which is assembled when needed. In the casewhere it is assembled when needed, the impregnation tank 1, the lidportion 2 for the impregnation tank 1, the introductory part 13 of theinlet 11, the introductory part 17 of he inlet 5, the introductory part18 of the outlet 6, the introductory part 19 of the inlet 8, theintroductory part 20 of the outlet 9, the medium 12 (may be moldedtogether with the introductory part 13 in advance) are providedseparately and connection of respective ones is achieved through tapermeans, for example.

As for the material of cartridge apparatus, a wide variety of knownplastic materials may be used.

The preparation of the present invention enables (1) administration withhigh quality for a long period of time without necessitating for lowtemperature transportation, such as an ultra-low temperature tank forstoring cell extracts for cell-free protein synthesis or using dry ice,etc. (2) By freeze-drying the cell preparation containing extracts forcell-free protein synthesis of the present invention, as it is, whichwas prepared by preliminarily mixing cell extracts for cell-free proteinsynthesis, amino acids, synthesis substrates such as ATP, and chemicalsubstances that are indispensable for translation reaction or increasethe efficiency of the reaction, such as various ions, the preparationcan be readily stored or transported in the form that retains highactivity of protein synthesis.

The preparation containing cell extracts for cell-free protein synthesisof the present invention does not need preparation of reaction mixtureupon ex vivo protein synthesis. According to the present invention,basically addition of only water and target translation templates (mRNA)provides means for identification of gene products, synthesis thereof ona large scale, or easy analysis of translation mechanism thereof.

The present invention enables stable storage and supply of substancesthat are unstable at normal temperature such as substances participatingin a cell-free protein synthesis reaction system, containing cellextracts for cell-free protein synthesis, other synthesis substratesexclusive of translation templates, energy sources, and chemicalsubstances that are indispensable for translation reaction or increasethe efficiency of the reaction, such as various ions, necessary for thecell-free protein synthesis system, and also enables administration ofsuch substances with high quality for a long period of time. Provisionof a freeze-dried preparation containing cell extracts for cell-freeprotein synthesis according to the present invention facilitateshandling of cell-free protein synthesis system as compared with theconventional handling and no necessity for preparing reaction mixtureshortens the operational process to improve usefulness of the cell-freeprotein synthesis system in industry.

Another means of the present invention utilizes the principle ofmolecular sieving as a carrier of a reaction tank for cell-free proteinsynthesis and enables a large volume cell-free protein synthesis thathas been difficult to be achieved by a conventional continuous cell-freeprotein synthesis method using a membrane.

Further, still another means of the present invention has led to theinvention of an automatic continuous protein synthesis apparatus of adialysis type to simplify the synthesis of protein.

EXAMPLES

Hereafter, the present invention will be described in further detail byexamples with reference to a preparation containing cell extracts forcell-free protein synthesis using wheat germ. However, the followingexamples should be construed obtaining concrete knowledge on the presentinvention and the scope thereof should by no means be limited by thefollowing examples.

Example 1

(Preparation of Wheat Germ Extracts)

As the method for isolating intact germ (having capability ofgermination) from seeds by use of milling, floatation and sieving, themethod of Johnston et al. (Johnston, F. B. et al. (1957) Nature, 179,160-161) was used with some modification. That is, Chihoku wheat seeds(non-sterilized) produced in Hokkaido were added to a mill (Rotor SpeedMill pulverisette Type 14) in a rate of 100 g per minute and pulverizedmildly at a rotation number of 8,000 rpm. The crushed seeds werepulverized again at 6,000 rpm and sieved to obtain a crude germ fraction(mesh size: 0.71 mm to 1.00 mm) and then germs having capability ofgermination were recovered by floatation using a mixed solution ofcarbon tetrachloride and cyclohexane (carbontetrachloride:cyclohexane=2.5:1) and the organic solution was removed bydrying at room temperature. Impurities such as seed coat contaminatingthe germ fraction was removed by adsorption using an electrified bodysuch as a polyethylene plate.

The germ particles were classified into three fractions of small (0.71mm to 0.85 mm), medium (0.85 mm to 1 mm), and light particle (0.85 mm to1 mm and light in weight) respectively, using a sieve and a staticelectricity and finally classified visually. The small particle fractionshowed the highest protein synthesis activity. As for the lightparticle, it is presumed that germs with a small injury caused at thepulverization underwent breakage which proceeded during the floatation.Then, to completely remove the wheat albumen component from thespecimen, the wheat germs were placed in a gauze bag and washed withcool distilled water (DDW) while cooling, suspended in a 0.5% NP-40,nonionic surfactant, solution, and washed repeatedly using anultrasonicator until the washings were not turbid in white any longer.After ultrasonic washing once again in the presence of distilled water,wheat germs were filtered by suction and the obtained wheat germs werewashed repeatedly in several times with cool distilled water (DDW) topurify the wheat germs.

The preparation of wheat germ extracts was performed in accordance withthe conventional method (Erickson, A. H. et al. (1996) Methods inEnzymol., 96, 38-50). The following operation was carried out at 4° C.The purified wheat germs frozen in a liquid nitrogen were pulverized ina mortar and to the powder obtained, an extraction solution was added 1ml/g of powder. Said solution contains 80 mM HEPES-KOH, pH 7.8, 200 mMpotassium acetate, 2 mM magnesium acetate, 4 mM calcium chloride, 8 mMdithiothreitol, each 0.6 mM of 20 kinds L-amino acids, each 1 μM of FUT,E-64 and PMSF, inhibitors for proteolytic enzymes and was obtained byPatterson et al. Method, with a partial modification. The solution wasstirred taking care so that no foam was generated. The supernatantobtained after centrifugation at 30,000 G for 15 minutes was recoveredas germ extracts and subjected to gel filtration using a Sephadex G-25column (Coarse) previously equilibrated with a solution (40 mMHEPES-KOH, pH 7.8, 100 mM potassium acetate, 5 mM magnesium acetate, and4 mM dithiothreitol). The concentration of the specimen was adjusted to170 to 250 A 260 nm (A260/A280=1.5).

The deadenylation ratio of the preparation, thus obtained was always 1%or less in plural practices in and results of 0.01 to 0.3% were obtained(the deadenylation ratio was measured according to an elimination methodwith aniline under acidic conditions: Endo, Y. et al. (1987) J. Biol.Chem., 262, 5908-5912, Yoshinari, S. et al. (1966) Eur. J. Biochem.,242, 585-591). The contanminants in tritin were assayed by an immunoblotmethod using an anti-tritin antibody, the contaminant showed below thedetection limit.

Example 2

(Continuous Wheat Germ Cell-Free Protein Synthesis by a Dialysis Method)

The method for continuous wheat germ cell-free protein synthesis hasbeen previously reported in the literature (Endo, Y. et al. (1992) J.Biotech., 25, 221-230). The reaction mixture obtained in Example 1, wascharged in a Dispo Dialyzer (Spectra/Por® CE, MWCO: 25 k, volume: 0.5ml) and the reaction was carried out at 20° C. in a dialysis systemagainst 10 folds volume of the reaction mixture of a dialysis externalliquid (20 mM HEPES-KOH, pH 7.6, 95 mM potassium acetate, 2.65 mMmagnesium acetate, 4 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, 16 mMcreatine phosphate, 0.38 mM spermidine, 20 kinds of L-type amino acids)(each 0.3 mM), 0.005% NaN₃, 0.05% NP-40, E-64, PMSF, each 1 mM).

Under the conditions obtained by the above method, continuous cell-freeprotein synthesis for wheat germ was tried and, as a result, variousproteins having molecular weights ranging from 5,000 to 130,000 could besynthesized efficiently and the efficiency of synthesis was 0.3 to 1.8mg per ml of the reaction volume (Table 1).

TABLE 1 Amount of synthesis of and properties of protein speciessynthesized under wheat germ cell-free system (dialysis system) (i) DHFR(20 kDa) 1.8 mg/ml (having activity) (ii) Human mitochondrial 1.3 mg/ml(having no activity, antigen Met-tRNA synthetase (84 kDa) for preparingan antibody) (iii) Luciferase (60 kDa) 0.7 mg/ml (having activity) (iv)Green fluorescent protein 0.4 mg/ml (having activity) (27 kDa) (v) HumanRNA helicase A 0.3 mg/ml (having no activity, an (130 kDa) antigen forpreparing an antibody) (vi) Proteasome Activator each 0.5 mg/ml (havingno activity, an Protein α, β, and γ antigen for preparing an antibody)(28, 28, and 31 kDa, respectively)

In the above examples, the method for assaying the protein synthesisactivity was performed in accordance with the method described in theprevious report by Endo et al. The method for measuring theincorporation of protein into ¹⁴C-leucine, the isolation of synthesizedprotein by SDS-polyacrylamide gel electrophoresis and dyeing withCoomassie Brilliant Blue, and polyribosome pattern assay method by asucrose density gradient centrifugation method were performed inaccordance with the papers by Endo et al. (Endo, Y. et al. (1992) J.Biotech., 25, 221-230, Endo, Y. et al. (1975) Biochim. Biophys. Acta,383, 305-315).

Example 3

(Forming Preparation from Wheat Germ Extracts)

The wheat germ extracts obtained by the method of Example 1, was frozenwith a liquid nitrogen and then water was removed therefrom in anordinary freeze-drying apparatus (Labconc Freeze Dry System Freezone4.5), which was operated for 3 hours. The powdery specimen thus preparedwas stored in tubes by two methods, i.e., sealed in vacuum and undernitrogen gas atmosphere, respectively.

Example 4

(Confirmation for the Effect of Protein Synthesis)

The wheat germ extracts-containing preparation according to the presentinvention, obtained by freeze-drying and preparing by the method ofExample 3 was stored at −80° C. for 2 months and separatelynon-freeze-dried wheat germ extracts were stored for 2 months in liquidnitrogen (−196° C.). Both wheat germ extracts were compared with eachother for protein synthesis activity.

To each wheat germ extract were added 30 mM HEPES-KOH, pH 7.6, 95 mMpotassium acetate, 2.65 mM magnesium acetate, 2.85 mM dithiothreitol,0.5 mg/ml creatine kinase, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatinephosphate, 0.380 mM spermidine, twenty kinds of L-type amino acids (each0.3 mM), as a composition in accordance with a method of Erickson etal., and separately after addition of 1,000 units/ml ribonucleaseinhibitor (RNasin) and NP-40 (final concentration: 0.05%), dihydrofolatereductase mRNA with a CAP (80 μg/ml of reaction volume) prepared by themethod previously reported by the present inventors, (Endo, Y. et al.(1992) J. Biotech., 25, 221-230) and 50 μCi (per ml of reaction volume)of [U-¹⁴C]-leucine (166 mCi/mmol) were added thereto. The proteinsynthesis activity was measured using the incorporation of[U-¹⁴C]-leucine as a marker. The reaction was carried out at 20 to 30°C.

As shown in FIG. 1, even after storage at a temperature of −80° C. for 2months, the containing preparation (▪--▪) freeze-dried wheat extractsretained protein synthesis activity, equivalent to that obtained by theconventional liquid nitrogen storage (□--□). Upon study on the storagetemperature after freeze-drying, comparing the protein synthesisactivity after a storage period of 1 month, −80° C. (--) was the moststable but when stored at 4° C. (▪--▪) or at room temperature (□--□) theprotein synthesis activity was retained sufficiently (FIG. 2).

That is, by applying the means of the present invention, the cellextracts for cell-free protein synthesis can be stored and supplied withmaintaining high quality at a temperature of −80° C. to roomtemperature, which is higher than the conventional storage temperature.

Example 5

(Forming Preparation Containing Wheat Germ Extracts for Cell-FreeProtein Synthesis)

The reaction mixture contained 20 to 60% by volume of the wheat germextracts prepared by the method of Example 1, to which were added 30 mMHEPES-KOH, pH 7.6, 95 mM potassium acetate, 2.65 mM magnesium acetate,2.85 mM dithiothreitol, 0.5 mg/ml creatine kinase, 1.2 mM ATP, 0.25 mMGTP, 16 mM creatine phosphate, 0.380 mM spermidine, 20 kinds of L-typeamino acids (each 0.3 mM), as a composition in accordance with themethod of Erickson et al. above, and then the mixture was frozen in aliquid nitrogen, followed by removing water in an ordinary freeze-dryingapparatus. The powdery specimen thus prepared was stored in tubes,sealed in vacuum or under nitrogen gas atomsphere so that the componentswill not undergo chemical reactions.

Example 6

(Measurement of Protein Synthesis Activity)

The preparation containing wheat germ extracts for cell-free proteinsynthesis prepared by the method of Example 5 according to the presentinvention was stored at −80° C. for 2 months and the wheat germ extractsfrom cell-free protein synthesis, in which the wheat germ extractsprepared by the conventional method and prepared in liquid nitrogen(−196° C.) for the same period of time as above, was added in substanceshaving the composition prepared in accordance with a method of Ericksonet al. stated in Example 5, were compared for their protein synthesisactivity. After addition of 1,000 units/ml ribonuclease inhibitor(RNasin) and NP-40 (final concentration: 0.05%), the measurement ofprotein synthesis activity was performed by the method of Example 4. Asshown in FIG. 3, the preparation containing freeze-dried wheat germextracts for cell-free protein synthesis of the present invention (□--□)retained protein synthesis activity equivalent to that of thepreparation prepared by the conventional method and stored in liquidnitrogen (▪--▪).

That is, by applying the means of the present invention, the preparationcontaining cell extracts for cell-free protein synthesis can be storedand supplied with maintaining high quality at a temperature of −80° C.to room temperature, which is higher than the conventional storagetemperature, and a method for simplifying the operational process forcell-free protein synthesis system can be provided.

Example 7

(Continuous Cell-Free Protein Synthesis using Gel Filtration ColumnChromatography as a Reaction Tank: Manual Open Column Method)

After swelling Sephadex G-25 (fine) manufactured by Pharmacia AB, as acarrier for gel filtration, the column was equilibrated with a buffersolution having the composition (comprising 20 mM HEPES-KOH, pH 7.6, 95mM potassium acetate, 2.65 mM magnesium acetate, 4 mM dithiothreitol,1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.380 mM spermidine,20 kinds of L-type amino acids (each 0.3 mM), 0.005% NaN₃, and 0.05%NP-40, E-64, PMSF (each 1 mM)), and packed in a column (10 mm in innerdiameter, 100 mm in length:).

To this column 0.1 ml of the cell-free protein synthesis reactionsolution prepared by the method of Example 1 (containing 80 μg per ml ofreaction mixture of dihydrofolate reductase mRNA with 5′-CAP as atranslation template) was overlaid and incubated at 23° C. for 1 hour,followed by supplying components necessary for the synthesis such asamino acids, energy sources, etc. at a flow rate of 0.5 ml per hourusing a peristalic pump. After 12 hours' reaction, 1 μl of the specimenwas subjected to 12.5% SDS-polyacrylamide gel electrophoresis to isolateprotein, which was dyed with Coomassie Brilliant Blue and further theamount of synthesized protein was determined using a densitometer (Endo,Y. et al., (1992) J. Biotech., 25, 221-230, Endo, Y. et al., (1975)Biochim. Biophys. Acta, 383, 305-315).

The results obtained are shown in FIG. 4A. Lane 1 shows the results inthe absence of templates, and Lane 3 shows the results of synthesis by acolumn method for 12 hours. The left-side Lane represents molecularweight markers of 94 kDa, 67 kDa, 43 kDa, 30 kDa, and 20.1 kDa,respectively from top. The arrow indicates the synthesized dihydrofolatereductase.

Example 8

(Continuous Cell-Free Protein Synthesis using a Gel Filtration ColumnChromatography as a Reaction Tank: Liquid Chromatography Apparatus)

The composition for reaction mixture, reaction method, and assay methodin Example 1 were all repeated except that a column (HR 10/30, innerdiameter: 10 mm, length: 300 mm) packed with Sephadex G-25 (fine)manufactured by Pharmacia AB as a carrier for gel filtration was used, aPharmacia Biotech SMART® System liquid chromatography apparatus wasutilized and the reaction volume was changed to 0.3 ml. The resultsobtained are shown as Lane 2 in Table 4B.

As Comparative Example, CONTINUOUS WHEAT GERM CELL-FREE PROTEINSYNTHESIS using a dialysis method was studied. The reaction mixture forcell-free protein synthesis prepared by the method of Example 1 wascharged in Dispo Dialyzer (Spectra/Por® CE, MWCO: 25 k, volume: 0.5 ml)and the reaction was carried out at 23 to 30° C. for 12 hours in adialysis system against 10 folds the volume of the reaction mixture of adialysis external liquid (comprising 20 mM HEPES-KOH, pH 7.6, 95 mMpotassium acetate, 2.65 mM magnesium acetate, 4 mM dithiothreitol, 1.2mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.380 mM spermidine, 20kinds of L-type amino acids (each 0.3 mM), 0.005% NaN₃, 0.05% NP-40,E-64, PMSF, each 1 mM). The results obtained are shown Lane 2 in FIG.4A, and Lane 1 in FIG. 4B.

As shown in FIG. 4, it has been experimentally confirmed that thedehydrofolate reductase used as a model can be produced by a columnmethod in a synthesis yield (0.25 mg/ml of reaction volume) similar tothat obtained in a dialysis method either by using a manual open columnmethod or liquid chromatography apparatus. An increase in column sizeenables production of protein on a large scale.

Example 9

(Improvement in the Synthesis Efficiency by Prolonging ReactionMaintenance Time by Supplemental Addition of mRNA and Creatine Kinase)

Using mRNA coding for dihydrofolate reductase as a model, synthesisreaction was performed for 24 hours. The reaction mixture contained 48%by volume of wheat germ extracts obtained in Example 1 and 1,000units/ml ribonuclease inhibitor (RNasin), 30 mM HEPES-KOH, pH 7.6, 95 mMpotassium acetate, 2.65 mM magnesium acetate, 2.85 mM dithiothreitol,0.5 mg/ml creatine kinase, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatinephosphate, 0.380 mM spermidine, twenty kinds of L-type amino acids (each0.3 mM), 0.05% NP-40 as a composition according to the method ofErickson et al., and in addition mRNA with a CAP (80 μg/ml reactionvolume), coding for dihydrofolate reductase prepared by the methodalready reported (Endo, Y. et al. (1992) J. Biotech., 25, 221-230).

The reaction mixture was allowed to react at 23° C. using a dialysissystem against 20 folds by volume of a dialysis external liquid(containing 20 mM HEPES-KOH, pH 7.6, 95 mM potassium acetate, 2.65 mMmagnesium acetate, 4 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, 16 mMcreatine phosphate, 0.380 mM spermidine, 20 kinds of L-type amino acids(each 0.3 mM), 0.005% NaN₃, and 0.05% NP-40, E-64, PMSF (each 1 mM)).

After 12 hours from the initiation of the reaction, 20 μg per ml ofreaction volume of mRNA with a CAP coding for dihydrofolate reductaseand 200 μg per ml of reaction volume of creatine kinase weresupplementally added. Separately, only 20 μg per ml of reaction volumeof mRNA with a CAP coding for dihydrofolate reductase is supplementallyadded, or alternately only 200 μg per ml of reaction volume of creatinekinase was supplementally added. As a comparative control, the reactionwas carried out without supplemental addition of either of thesubstances. The operation was performed manually and dialysis externalliquid was not changed. The mass of synthesized protein was obtained bysubjecting the obtained protein to SDS-polyacrylamide gelelectrophoresis and dyeing thereof with Coomassie Brilliant Blue,measuring the intensity of the dyed band and calculating the ratio ofthe intensity of dyed band to that of standard preparation (Endo, Y. etal. (1992) J. Biotech., 25, 221-230, Endo, Y. et al. (1975) Biochim.Biophys. Acta, 383, 305-315).

As shown in FIG. 7, it can be seen that by the supplemental addition ofmRNA and creatine kinase (o-o) after 12 hours from the initiation ofreaction, the reaction lasted substantially linearly. In the case ofsupplemental addition of either one of them (Δ-Δ) or (∇-∇), the reactionwas terminated similarly to the case where no supplemental addition wasmade (□-□). That is, here, the above-indicated possibilities(termination of protein synthesis reaction due to a decrease in templateactivity of mRNA and exhaustion of energy sources resulting from adecrease in creatine kinase activity) were experimentally confirmed anda solution therefor was completed.

Example 10

(Improvement in Synthesis Efficiency for Prolonging Reaction MaintenanceTime by Chronological Exchanging Dialysis External Liquid)

Protein synthesis was performed for 60 hours using mRNA coding fordihydrofolate reductase as a model by setting the conditions forcomposition of reaction mixture, temperature, etc. to the same as inExample 9 and the dialysis external liquid was exchanged after 24 hoursand 45 hours, respectively, after the initiation of the reaction. Ascomparative control, protein synthesis was carried out without changingthe dialysis external liquid. In either of the reaction systems, 20 μgper ml reaction volume of mRNA with a CAP coding for dihydrofolatereductase and 200 μg per ml reaction volume of creatine kinase weresupplementally added. The mass of synthesized protein was measured bythe method of Example 9.

As shown in FIG. 8, the synthesis proceeded substantially linearlywithout exchange of the dialysis external liquid (Δ-Δ) up until 24hours. However, the reaction rate was decreased extremely in about 30hours. However, the exchange of dialysis external liquid (o-o) in every24 hours (arrows) enabled the protein synthesis to last for at least 60hours. That is, the above-described possibilities (termination ofprotein synthesis reaction due to the exhaustion of raw materialsessential for the protein synthesis and the accumulation of by-products)were experimentally confirmed and a method for solving them wascompleted.

Example 11

(Prolongation of Reaction Maintenance Time and Improvement in theSynthesis Efficiency of by Automatic Supplemental Addition of mRNA andCreatine Kinase and Automatic Exchange of a Dialysis External Liquidusing an Automatic Apparatus for Continuous Cell-Free Protein Synthesis)

Protein synthesis was performed for 60 hours using mRNA coding fordihydrofolate reductase as a model by setting the conditions ofcomposition of reaction mixture, temperature, etc. to the same as inExample 9 and using an automatic apparatus for continuous cell-freeprotein synthesis. In every 12 hours after the initiation of thereaction, 20 μg per ml reaction volume of mRNA with a CAP coding fordihydrofolate reductase and 200 μg per ml reaction volume of creatinekinase each in an amount of 5 μl separately stored at 4° C. weresupplementally added and allowed to react. The dialysis external liquidwas supplied (0.3 ml/hour) continuously from a storage vessel maintainedat 4° C. to a dialysis vessel, and discharged from the dialysis vesselat the same flow rate. After subjecting 1 μl equivalent amount, to theinitial amount of reaction mixture (0.5 ml), of the reaction mixture toSDS-polyacrylamide gel electrophoresis, the protein was dyed withCoomassie Blue (FIG. 9(A)). As comparative control, the synthesis ofprotein was carried out in the same manner as described above aftersupplemental addition of only creatine kinase and without supplementaladdition of mRNA with a CAP coding for dihydrofolate reductase (FIG.9(B)). Also, to 1 μl of specimen after 0 hour from the initiation ofreaction was added 1 μg of dihydrofolate reductase standard product andthe electrophoresis and dyeing were performed in the same manner asdescribed above (right side Lane in FIG. 9(A)). The amount ofsynthesized protein was measured by the method described in Example 9(FIG. 9(C)).

As shown in FIG. 9, in the case where no supplemental addition of mRNAwith a CAP coding for dihydrofolate reductase was performed butsupplemental addition of only creatine kinase was performed (Δ-Δ), thereaction was terminated in about 15 hours. Though not shown in thedrawings, similar termination of reaction was observed in the case wherecreatine kinase was not supplemented but only mRNA with a CAP coding fordihydrofolate reductase was supplemented. On the other hand, automaticsupplement of mRNA with a CAP coding for dihydrofolate reductase and ofcreatine kinase for every 12 hours and automatic exchange of dialysisexternal liquid after every 24 hours (o-o) enabled the cell-free proteinsynthesis to automatically proceed efficiently. That is, it wasconfirmed that the apparatus of the present invention functionedeffectively.

1. A preparation comprising a cell extract from the germ of floweringplants for cell-free protein synthesis prepared by substantiallycompletely free of endosperm from said cell extract, therebysubstantially excluding the systems involved in inhibiting the cellextract's protein synthesis reactions.
 2. The preparation according toclaim 1, wherein said systems involved in inhibiting the cell extract'sprotein synthesis reactions are substantially excluded by treating saidcell extract with a nonionic surfactant.
 3. The preparation according toclaim 2, wherein the cell extract is further treated by usingultrasonication with said surfactant.
 4. The preparation according toclaim 3, wherein the excluding of said systems involved in inhibitingthe cell extract's protein synthesis reactions serves to controldeadenylation of ribosome.
 5. The preparation according to claim 3,wherein said preparation can be stored in room temperature and maintainsbiological functions of said cell extract.
 6. The preparation accordingto claim 2, wherein the excluding of said systems involved in inhibitingthe cell extract's protein synthesis reactions serves to controldeadenylation of ribosome.
 7. The preparation according to claim 2,wherein said preparation can be stored in room temperature and maintainsbiological functions of said cell extract.
 8. The preparation accordingto claim 1, wherein the excluding of said systems involved in inhibitingthe cell extract's protein synthesis reactions serves to controldeactivation of ribosomes present in said cell extract.
 9. Thepreparation according to claim 1, wherein a substance is present tocontrols deadenylation of ribosomes and to exclude systems involving theinhibition of protein synthesis.
 10. The preparation according to claim9, wherein said preparation can be stored in room temperature andmaintains biological functions of said cell extract.
 11. The preparationaccording to claim 1, wherein the cell extract is from an embryo andsaid embryo is treated by adding nonionic surfactant and a substancecontrolling deadenylation of ribosome to exclude systems involving theinhibition of protein synthesis.
 12. The preparation according to claim11, wherein said preparation can be stored in room temperature andmaintains biological functions of said cell extract.
 13. A Thepreparation according to claim 1, wherein said preparation can be storedin room temperature and maintains biological functions of said cellextract.
 14. The preparation according to claim 13, wherein thepreparation is in dried form.
 15. The preparation according to claim 14,wherein the preparation is formulated by freeze-drying.
 16. Thepreparation according to claim 1, further comprising a synthesizedsubstrate, an amino acid, an energy source, a surfactant, an ioniccompound, or combinations thereof, wherein said preparation can bestored in room temperature and maintains biological functions of saidcell extract.
 17. A preparation containing a cell extract for cell-freeprotein synthesis, comprising an extract of wheat embryo obtained bywashing the wheat embryo with nonionic surfactant to completely removeany endosperm contaminants from the wheat embryo, wherein thedeadenylation rate of the wheat extract is 1% or lower, and the drypreparation of the wheat embryo extract maintains stability at roomtemperature; and wherein said wheat extract is used in a continuouscell-free protein synthesis involving a replenishment of the substrateand other substances for protein synthesis, and the synthesis showsconstant performance even in 24^(th) hour after starting the synthesisand shows at least 1 mg/ml or higher in synthesis level in said 24^(th)hour.
 18. A method for synthesizing a protein in a cell-free systemwhich is capable of recovering the synthesized protein, said methodcomprising the steps of providing a reaction vessel containing rawmaterial substances that participate in cell-free protein synthesis,wherein the raw material substances comprise the preparation of claim 1,and wherein the reaction vessel comprises a carrier capable of molecularsieving, carrying out cell-free protein synthesis to obtain asynthesized protein, during which synthesis the synthesized productprotein is separated from the raw material substances by moving throughthe carrier, and recovering the separated protein.
 19. A method forsynthesizing a protein in a cell-free system which is capable ofrecovering the synthesized protein, said method comprising the steps ofproviding a reaction vessel containing raw material substances thatparticipate in cell-free protein synthesis, wherein the raw materialsubstances comprise the preparation of claim 1, and wherein the reactionvessel comprises a dialysis membrane that separates the reaction vesselinto a reaction phase and an external phase, carrying out cell-freeprotein synthesis, during which synthesis the synthesized protein isproduced in the reaction phase and is separated from the raw materialsubstances through the dialysis membrane, and recovering the separatedprotein.
 20. A method of synthesizing a protein in a cell-free systemcomprising the steps of providing raw material substances thatparticipate in cell-free protein synthesis, wherein the raw materialsubstances comprise the preparation of claim 1, and carrying outcell-free protein synthesis in which the raw material substancesparticipate to produce a synthesized protein.
 21. A method ofsynthesizing a protein in a cell-free system comprising the steps ofproviding raw material substances that participate in cell-free proteinsynthesis, wherein the raw material substances comprise the preparationof claim 2, and carrying out cell-free protein synthesis in which theraw material substances participate to produce a synthesized protein.22. A method of synthesizing a protein in a cell-free system comprisingthe steps of providing raw material substances that participate incell-free protein synthesis, wherein the raw material substancescomprise the preparation of claim 3, and carrying out cell-free proteinsynthesis in which the raw material substances participate to produce asynthesized protein.
 23. A method of synthesizing a protein in acell-free system comprising the steps of providing raw materialsubstances that participate in cell-free protein synthesis, wherein theraw material substances comprise the preparation of claim 8, andcarrying out cell-free protein synthesis in which the raw materialsubstances participate to produce a synthesized protein.
 24. A method ofsynthesizing a protein in a cell-free system comprising the steps ofproviding raw material substances that participate in cell-free proteinsynthesis, wherein the raw material substances comprise the preparationof claim 9, and carrying out cell-free protein synthesis in which theraw material substances participate to produce a synthesized protein.25. A method of synthesizing a protein in a cell-free system comprisingthe steps of providing raw material substances that participate incell-free protein synthesis, wherein the raw material substancescomprise the preparation of claim 11, and carrying out cell-free proteinsynthesis in which the raw material substances participate to produce asynthesized protein.
 26. A method of synthesizing a protein in acell-free system comprising the steps of providing raw materialsubstances that participate in cell-free protein synthesis, wherein theraw material substances comprise the preparation of claim 17, andcarrying out cell-free protein synthesis in which the raw materialsubstances participate to produce a synthesized protein.